Inducible depletion of adult skeletal muscle stem cells impairs the regeneration of neuromuscular junctions

1Skeletal muscle is composed of long multinucleated cells, muscle fibers (myofibers), which function as primary effectors for force production and contribute to the regulation of whole body metabolism. Although primarily a post-mitotic tissue, adult skeletal muscle possesses a remarkable capacity for regeneration. This capacity is endowed by a population of resident stem cells, satellite cells (SCs), identified by the expression of the paired box transcription factor Pax7. In adults, SCs normally reside in a quiescent state at the interface between the myofiber and overlying basal lamina. However, in response to degenerative stimuli, Pax7+ SCs activate and divide to produce myogenic progenitors for skeletal muscle regeneration. Since the initial identification of SCs over 50 years ago, many studies have examined roles for these cells in a plethora of distinct models of skeletal muscle injury, adaptability and disease. Both depletion of Pax7+ SCs and targeted disruption of Pax7 have shown these cells to be an essential source of myonuclei for skeletal muscle regeneration. Depletion studies have revealed roles for Pax7+ SCs in late stages of experimentally induced skeletal muscle hypertrophy. Recently, SCs were also shown to function as a source of growth factors to facilitate bone fracture healing. Similar strategies have shown SCs contribute to myofibers and regulate aspects of skeletal muscle integrity during aging.

2In adults, each myofiber is innervated by a single axon from a motor neuron. Innervation occurs at a specialized site in the central region of myofibers, the neuromuscular junction (NMJ). The NMJ, which occupies approximately 0.1% of the surface area of a myofiber, initiates action potential propagation required for excitation/contraction coupling to generate force for movement and maintain myofiber properties. Consistent with a vital role for NMJ integrity in skeletal muscle maintenance, neuromuscular disruptions elicit severe myofiber atrophy, and are frequently associated with skeletal muscle dysfunction observed in the context of neuromuscular diseases (NMDs) and aging.

3The regeneration of NMJs in response to peripheral nerve lesions can occur, however the length and quality of recovery depends on the severity of injury. Remarkably, the initial reinnervation of synaptic basal lamina by motor axons can proceed in the absence of myofibers. However, in the absence of myofibers and associated SCs, the continued maintenance of reinnervated NMJs on basal lamina ghosts eventually declines. These observations indicate that myofiber derived factors or associated SCs may be required for the continued differentiation and maintenance of regenerated NMJs. Accordingly, myofiber components and derived factors have been identified to assist in the progressive differentiation of developing and regenerating NMJs. In models of chronic denervation, where reinnervation is prevented, SCs activate and divide; however, the derived progenitors migrate into interstitial spaces, undergo defective differentiation or are lost via apoptosis. Furthermore, little turnover of myonuclei and fusion of myogenic progenitors to chronically denervated parent myofibers have been observed. Collectively, these studies have suggested SCs have limited, if any, roles in the regeneration of NMJs upon neuromuscular disruptions.

4Through the use of targeted genetic strategies we sought to reexamine the fates and roles of SCs in a model of peripheral nerve injury that enables NMJ regeneration. In this study we find a limited proportion of SCs activate and divide during NMJ reestablishment. Remarkably, while SC depletion did not lead to additional loss of skeletal muscle mass, it was sufficient to reduce myofiber size, increase inter-myofiber connective tissue (MCT) accumulation, and aggravate myofiber type transitions connected to NMJ disruption. These phenotypes were associated with further declines in force generation capacity. Examination of fate revealed increased SC activity and fusion of indelibly labeled SC-derived progenitors to myofibers predominantly in the vicinity of regenerating NMJs. Consistent with a role for SCs in the regeneration of NMJs, we found that SC depletion led to deficits in NMJ reinnervation, reductions in post-synaptic morphology and loss of post-synaptic myonuclei. Collectively our findings reveal fates and roles for SCs in the regeneration of NMJs and regulation of skeletal muscle integrity upon neuromuscular disruption.

5To examine the roles of SCs in skeletal muscles upon neuromuscular disruption we generated Pax7CreER/+; Rosa26DTA/+ (P7DTA) and Pax7+/+; Rosa26DTA/+ (Ctrl) mice. These mice enable tamoxifen (Tmx)-mediated expression of diphtheria toxin-A (DTA) to deplete Pax7+ SCs to levels that prevent the regeneration of skeletal muscle. We employed 1-2 mm sciatic nerve transection (SNT) to disrupt lower-limb NMJs. This form of surgery leads to complete denervation of adult NMJs. Although delayed, reinnervation as assessed by immunofluorescence (IF) and physiological measures does occur 4–6 weeks after SNT. Consistent with previous reports, a modest albeit significant increase in Pax7+ SC number was observed 6 weeks after SNT surgery (Figure 1). After Tmx administration, extensive depletion of Pax7+ SCs occurred regardless of sham or SNT surgery (Figure 1).

6We assessed various morphological parameters to determine the consequences of SC depletion on SNT-induced skeletal muscle atrophy. Gross examination of TA skeletal muscles 6 weeks after SNT surgery revealed overall reductions in girth, which were not noticeably different between P7DTA and Ctrl (Figure 2A). While the mass of TA, extensor digitorum longus (EDL) and soleus muscles 6 weeks after SNT relative to contralateral sham controls remained low, SC depletion did not induce further loss of muscle mass (Figure 2B–D). Examination of individual myofibers from EDL muscles that had been fixed prior to isolation from lower limbs revealed only modest reductions in myonuclear density after SNT and SC depletion (Figure 2—figure supplement 1A and C). Based on these moderate losses, a small albeit significant level of myonuclear turnover was observed in P7DTA sham muscles. Therefore, the magnitude of myonuclear loss after SNT relative to sham in P7DTA skeletal muscles is small (Figure 2—figure supplement 1C). Next we examined myofiber size based on Laminin IF analysis of transverse sections from TA muscles (Figure 2E). Despite 6 weeks of recovery, Ctrl myofiber size after SNT remained ∼25% lower relative to contralateral sham, however, SC depletion led to further SNT-induced myofiber atrophy (Figure 2F). Distribution analysis of myofiber size did not reveal any significant differences between Ctrl and P7DTA muscles after sham surgery, but a significant shift towards smaller sizes after SNT surgery was observed upon SC depletion (Figure 2G). No change in myofiber numbers was observed after SNT and SC depletion (Figure 2—figure supplement 1B). Therefore, SC depletion did not lead to a significant change of overall muscle morphology and mass, but aggravated SNT-induced myofiber atrophy.

7Previous studies on rodent models or human patients reveal that chronic denervation, aging of skeletal muscles or NMDs can induce an increase in MCT content, an indicator of fibrosis. Also, SC depletion has been shown to result in extracellular matrix accumulation in the context of skeletal muscle regeneration, functional overload-induced hypertrophy and aging. Therefore, to determine if elevated MCT is associated with reductions in myofiber size upon SC depletion and SNT, we performed hematoxylin and eosin (H&E) and Sirius Red staining for collagens (Figure 3A,B). Surprisingly, although SNT surgery alone did not increase MCT content, when combined with SC depletion, a significant increase in fibrosis was observed (Figure 3C). Therefore, the lack of change in the mass of SC-depleted skeletal muscles relative to Ctrl after SNT surgery was accompanied by both increased fibrosis and myofiber atrophy.

8Most skeletal muscles are composed of heterogeneous mixtures of functionally distinct types of myofibers that differ in many parameters. Based on the expression of skeletal muscle myosin heavy chain (MyHC), different types of myofibers can be classified along a continuum: type I, IIA, IIX and IIB. In this regard, type I fibers are characterized by slower contraction kinetics and lower force generation, whereas at the other end of the continuum type IIB fibers exhibit faster contraction kinetics and greater force generation. Consistent with the vital influence of neural activity on myofiber type, myofibers innervated by the same motor neuron primarily express one MyHC isoform, and cross-innervation can induce the expression of isoforms indicative of the foreign nerve. Neuromuscular disruption is frequently associated with abnormal profiles of MyHC isoform expression within a given skeletal muscle. A typical feature of NMD and aging skeletal muscle is an increased occurrence of hybrid myofibers whereby two or more MyHC isoforms are co-expressed. To examine hybrid myofibers, we immuno-stained Ctrl and P7DTA inner TA/EDL muscle sections 6 weeks after sham or SNT surgery with antibodies that specifically detect MyHC IIX or all MyHCs except IIX, therefore hybrid IIX myofibers will be labeled with both. Very few MyHC IIX hybrid myofibers were found in adult inner TA/EDL muscles after sham surgery regardless of genotype (Figure 4A,B). Elevations in the proportion of hybrid myofibers were observed in Ctrl inner TA/EDL muscles 6 weeks after SNT, a phenotype exacerbated by SC depletion (Figure 4A,B). Assessment of inner TA/EDL muscles after SNT with antibodies specific for MyHC I, MyHC IIA and MyHC IIB revealed: i) very little if any expression of MyHC I; ii) inductions of MyHC IIA that were aggravated upon SC depletion; and iii) loss of MyHC IIB (Figure 4A and C–E). Together, these data indicate that the majority of hybrid myofibers observed after SNT are presumably type IIA/IIX.

9Modulations in factors that influence force transmission and excitation/contraction coupling, such as increased MCT and abnormalities in the expression of contractile elements can hinder intrinsic whole skeletal muscle contractile function. Since we observed inductions in MCT and deviations in the expression of MyHC isoforms, major components of the contractile apparatus, after SC depletion and SNT, we measured ex vivo muscle contractility of Ctrl and P7DTA EDL muscles. Initially we examined the time taken by a given muscle to reach peak tension upon stimulation at 150 Hz, a frequency that elicited peak tetanic contractile force. A similar lengthening in time to peak tension (TTP) was observed in Ctrl and P7DTA EDL muscles after SNT (Figure 5A, and Figure 5—figure supplement 1B). Although lengthening of TTP 6 weeks after SNT is consistent with shifts in MyHC expression with slower contractile character, SC depletion did not further influence this property. Next we examined the max tetanic force generated by Ctrl and P7DTA EDLs 6 weeks after SNT surgery upon stimulation at frequencies of increasing intensity. We found significant deficits in peak absolute and specific force generation in P7DTA EDL muscles in comparison to Ctrl after SNT at progressively higher frequencies (Figure 5B,C, and Figure 5—figure supplement 1A). Therefore, the exacerbated deficits in skeletal muscle integrity such as myofiber atrophy, increased MCT content and abnormal profiles of MyHC expression upon SC depletion and SNT were accompanied by declines in whole skeletal muscle force generation.

10Initially to assess SC fate we employed an in vivo BrdU incorporation assay to examine SNT-induced SC activation and division. Consistent with limited SC activation, only a fraction (∼25%) of the SC pool in TA muscles had incorporated BrdU after SNT (Figure 6A,B). To further examine the fate of Pax7+ SCs in response to neuromuscular disruption, we generated a SC specific Pax7CreER/+; Rosa26mTmG/+ (P7mTmG) transgenic mouse line. The P7mTmG mouse ubiquitously expresses a loxP flanked membrane tomato red fluorescent reporter (RFP) that undergoes Tmx mediated excision to indelibly label Pax7+ SCs and derived cells with membrane GFP (GFP), enabling lineage tracing of SCs in skeletal muscles and individual myofibers after SNT. To initially characterize the efficiency of the P7mTmG line, we assessed GFP label in SCs and myofibers 24 hr after the last Tmx administration. We found that 24 hr after the last Tmx administration, >90% of the SC pool was GFP+, whereas myofibers were devoid of GFP label (Figure 7—figure supplement 1). Next, we examined SC derived GFP labeling after sham or SNT surgery. In comparison to sham, a marked increase in GFP + myofibers was observed in transverse sections from the mid belly of TA and EDL muscles after SNT (Figure 7A,B). Although these data indicate a high proportion of myofibers within TA and EDL muscles have undergone a fusion event after SNT, this activity likely reflects limited fusion within a given myofiber consistent with modest losses of myonuclei within P7DTA myofibers (Figure 2—figure supplement 1A,C). The occurrence of central nucleated myofibers (CNF), an indicator of degenerative/regenerative events of myofibers, was minimal and cannot explain the observed induction of GFP + myofibers after SNT (Figure 7—figure supplement 1C).

11Myofibers are long multinucleated cells, along which local events of turnover and gene activity can occur. To determine if SNT leads to regional activity and fusion of indelibly labeled SCs and derived progenitors, we isolated EDL single myofibers by collagenase digestion and processed them for the detection of post-synaptic acetylcholine receptors (AChRs) with fluorochrome-conjugated α-bungarotoxin (Btx) and GFP. We scored the GFP labelling based on location: i) ‘whole’ for GFP throughout the myofibers; ii) ‘end’ for GFP at myofiber ends/tips; iii) ‘middle’ (Mid) for central regions of myofibers where NMJs are located; and iv) ‘RFP’ for no GFP labelling (examples for RFP, End and Mid in Figure 7C,D). After sham surgery, only a small fraction of myofibers were GFP positive; none of these fibers had GFP throughout, and most labels were found either at the ends or middle regions, the latter being where NMJs are located (Figure 7E). After SNT surgery a greater proportion of myofibers were GFP positive, indicating the activation of at least some SCs and fusion of SC derived progenitors to myofibers (Figure 7E). In addition, the majority of the GFP label found in myofibers isolated from EDL muscles after SNT was located in the middle, within the vicinity of NMJs (Figure 7E). Therefore, neuromuscular disruption led to regional SC derived contributions along the length of a myofiber in proximity to the NMJ.

12Due to the regionalized response of SCs along myofibers near the NMJ after SNT, we employed P7DTA mice to test the importance of this local SC activity to NMJ regeneration upon neuromuscular disruption. Confocal IF microscopy and 3-D image analysis with Amira were used to assess NMJ regeneration. NMJs were identified with post-synaptic (AChR labeled with Btx) and pre-synaptic (SV2, Syt-2 and neurofilament) markers (Figure 8A). Initially, we examined the extent of reinnervation, defined as the coverage of post-synaptic regions by pre-synaptic markers. We considered a NMJ to be: i) innervated, if the vast majority of post-synaptic regions are covered by pre-synaptic terminal markers; ii) partially denervated, if > 5 μm length of an AChR enriched branch within the post-synaptic apparatus is not covered by pre-synaptic terminal markers while the other parts of the apparatus are; and iii) fully denervated, if > 90% of the post-synaptic apparatus is devoid of pre-synaptic nerve terminal markers. Consistent with previous reports, a large increase in partially/fully denervated NMJs in Ctrl TA muscles was observed after SNT compared to sham surgery, suggesting incomplete regeneration of NMJs (Figure 8B). Examination of P7DTA NMJs revealed that SC depletion was associated with a significantly higher proportion of NMJs that remained partially/fully denervated 6 weeks after SNT surgery (Figure 8B).

13Post-natal NMJ maturation is associated with post-synaptic morphological transitions from a plaque-like to a pretzel-like shape due to the formation of post-synaptic membrane invaginations (junctional folds). Aged and NMD-afflicted skeletal muscles are frequently associated with NMJ degeneration, manifested by loss of elaborate AChR-enriched branches and the characteristic pretzel-like morphology of mature adult NMJs. Because post-synaptic morphology is linked to NMJ integrity and disease, we measured the occurrence of NMJ degeneration in Ctrl and P7DTA skeletal muscles after sham or SNT surgery. We considered an NMJ to be plaque-like, or degenerated, if the AChR-enriched area resembled a patch devoid of defined elaborate branches >5 μm in length. Very few degenerated NMJs were found in Ctrl or P7DTA TA muscles 6 weeks after sham surgery regardless of genotype (Figure 8C). After SNT surgery there was a marked increase in the proportion of degenerated NMJs, a percentage that was substantially elevated by ∼ 25% with SC depletion (Figure 8C).

14Enriched at most adult NMJs are clusters of post-synaptic myonuclei specialized for the expression of genes required for AChR consolidation and the differentiation of pre-synaptic nerve terminals. Since SCs function as an essential source of myonuclei for skeletal muscle regeneration, local SC activity along denervated myofibers may be required for the maintenance of post-synaptic myonuclei. Therefore, we quantified the number of post-synaptic myonuclei (>25% DAPI covered by the Btx + post-synaptic apparatus) in TA muscles. Assessment of Ctrl and P7DTA NMJs 6 weeks after sham surgery did not reveal any significant alteration in the distribution of NMJs based on post-synaptic myonuclei size (Figure 8D). Also, no significant loss of post-synaptic myonuclei was found at Ctrl NMJs 6 weeks after SNT (Figure 8D). However, partially/fully denervated or degenerated (based on post-synaptic morphology, plaque-like) Ctrl NMJs had significantly fewer post-synaptic myonuclei, indicating that loss of post-synaptic myonuclei is a feature of NMJ degeneration (Figure 8—figure supplement 1). Consistent with a higher proportion of degenerated NMJs, a significant shift towards smaller post-synaptic myonuclear size was observed at P7DTA NMJs 6 weeks after SNT (Figure 8D). Collectively these data indicate that SC depletion, although not sufficient alone to trigger loss of NMJ integrity at homeostasis, did notably impair NMJ regeneration in response to neuromuscular disruption.

15In this report we interrogated the roles and fates of SCs in a model of neuromuscular regeneration. In doing so we found that upon NMJ disruption: i) SC depletion exacerbates myofiber atrophy and type transitions; ii) SC depletion leads to elevated fibrosis and declines in muscle force generating capacity; iii) SC-derived contributions prevail in the vicinity of NMJs; and iv) SC depletion leads to deficits in skeletal muscle reinnervation, reductions in post-synaptic morphology and loss of post-synaptic myonuclei. Through these findings we propose a cellular mechanism whereby SCs contribute to the regeneration of NMJs and skeletal muscle maintenance upon neuromuscular disruption. Based on the results presented here, an aspect of NMJ regeneration includes local SC activation, derived progenitor fusion and turnover of post-synaptic myonuclei.

16Although we did not observe any major alterations in whole muscle mass after SNT, SC depletion did lead to further declines in myofiber size and increased MCT accumulation. The connection between SC depletion and MCT accumulation or fibrosis has also been observed by other studies in the context of skeletal muscle regeneration, functional overload-induced hypertrophy and aging. A study by the Kardon group highlighted that MCT fibroblasts are dynamically regulated by SCs, and the positive feedback between SCs and fibroblasts ensures efficient and effective skeletal muscle regeneration. Results from the Peterson group revealed that SC depletion led to a fibrotic response in long-term overloaded muscles and elevated MCT accumulation in aged muscles, suggesting that SCs regulate the myofiber extracellular environment through inhibiting fibroblast function during skeletal muscle remodeling. In addition, multiple populations of muscle interstitial cells (MICs) have been identified with fibro-adipogenic potential, such as Fibro-adiopogenic progenitors (FAPs), PDGFRα+ MICs or ADAM12 + perivascular cells. These cells may serve as functional niche components for SCs, and their cross-talk with SCs may regulate muscle regeneration and fibrosis associated with NMDs. How the functional interactions between SCs, fibroblasts and MICs affect fibrosis either through cell contact or the release of soluble factors requires further study.

17Myofiber type transition and declines in force generation capacity are highly correlated with NMJ disruption. Rodent models revealed that peripheral nerve lesions cause fast to slow myofiber transitions and a substantial increase in the proportion of type IIA myofibers. A preferential loss of fast-type type IIB neuromuscular synapses has been reported in aged and NMD-afflicted muscles. Consistent with previous studies, we showed an induction of type IIA and loss of type IIB myofibers after SNT. This SNT-induced increase of type IIA myofibers was aggravated upon SC depletion, in line with our later finding that more P7DTA NMJs remain denervated after SNT. Concomitantly, after SNT and SC depletion we observed a notable induction of hybrid myofibers, a typical feature of NMD-afflicted and aged skeletal muscle. The mechanisms responsible for the occurrence of MyHC coexpression are still elusive. One possibility is induction of multiple MyHC isoforms following denervation and reinnervation by multiple motor neurons that specify distinct myofiber types. Our results also revealed that SC depletion led to declines in force generation capacity after neuromuscular disruption. Both accumulated MCT content and abnormal expression of contractile proteins could partially account for the loss of whole skeletal muscle force generation after SC depletion and SNT. Also, impaired NMJ regeneration in response to neuromuscular disruption might contribute to the additional loss of force generating capacity in SC-depleted muscles compared to Ctrl after SNT. In support, declines in force generating capacity are a feature of partially denervated and aged skeletal muscles.

18Limited if any cellular turnover and no net loss of myonuclei has been reported in chronically denervated skeletal muscle. Therefore, it has been concluded that muscle mass loss in the context of denervation-induced atrophy primarily reflects imbalances between protein synthesis and degradation as opposed to cellular turnover. Our SNT surgery, however, leads to complete denervation and the eventual reinnervation of the vast majority of myofibers 4–6 weeks after surgery. Chronic denervation models do have greater levels of myofiber atrophy than observed with the SNT employed here. Although chronic denervation leads to SC activation, it is generally accepted that limited fusion of SC derived progenitors to atrophying parent myofibers occurs. Rather, SCs and derived progenitors tend to migrate into interstitial spaces where they undergo apoptosis, are believed to contribute to the formation of nascent myofibers with distinct basal lamina juxtaposed to parent atrophying myofibers, or contribute to other forms of failed myogenesis. It will be of interest to determine whether SCs are a source of nascent myofibers and failed myogenesis, and how this contributes to myofiber size changes during chronic denervation.

19Even though only a small percentage of SCs were found to activate after SNT, surprisingly we found that SC activation and SC-derived progenitor fusion primarily occurred in central portions of myofibers, in the vicinity of NMJs. Moreover, more denervated and degenerated (based on post-synaptic morphology) NMJs observed in SC depleted skeletal muscles after SNT were associated with significant declines in the number of post-synaptic myonuclei at NMJs. These phenotypes indicate the importance of regionalized SC activity for regenerating NMJs. Central nucleation was not a prominent feature of myofibers 4–6 weeks after SNT. Recently transient central nuclei were observed along the length of aging myofibers at random locations. Whether such transient degenerative regenerative events occur in the vicinity of NMJs in response to SNT as part of program of myogenic progenitor differentiation and fusion remains to be determined. The factors that control SC activity and derived progenitor fusion at central myofiber regions in the vicinity of NMJs during NMJ disruption are unknown. One possibility is the local expression of SC fate regulators that support progenitor activity and fusion from myofibers, the principal SC niche cell. Alternatively, denervation may trigger the expression of factors that suppress SC activation and differentiation at extra-synaptic myofiber regions. Factors implicated in SC quiescence and activation include loss of Notch signaling and increased fibroblast growth factor (FGF) or hepatocyte growth factor (HGF)-induced receptor tyrosine kinase signaling. TGFβ superfamily signaling, which is elevated in denervated skeletal muscle, is a well-established suppressor of myogenic differentiation. In addition, Jak/Stat and Wnt signaling are factors that regulate SC derived myogenic progenitor differentiation. Whether any of these factors limit SC activation and differentiation in the vicinity of denervated NMJs will require molecular dissection of myofibers at distinct regions.

20Post-synaptic myonuclei are specialized for the expression of synapse-enriched genes required for the development, differentiation, consolidation and maintenance of both pre- and post-synaptic components. Consistent with vital roles, the etiology of some NMDs includes reductions in post-synaptic myonuclear number and integrity. Emery-Dreifuss muscular dystrophy is characterized by a loss of post-synaptic myonuclei together with deficits in skeletal muscle innervation, post-synaptic AChR morphology and the induction of gene expression programs consistent with denervation. A feature of Slow-channel syndrome, a congenital myasthenia disorder, includes apoptotic activity and the accumulation of DNA damage at post-synaptic myonuclei. Genetic studies have also shown the importance of post-synaptic myonuclei towards NMJ development. Mice null for Syne-1, a nuclear anchoring protein, display loss of post-synaptic myonuclei together with gross deficits in the innervation of embryonic skeletal muscles. Therefore, reductions of post-synaptic myonuclei in SC-depleted skeletal muscles after SNT could manifest in the loss of gene expression programs required for the regeneration and maintenance of NMJs.

21Similar to what we observed with SC-depleted skeletal muscles after NMJ disruption, myofiber type transitions, decreases in NMJ regenerative capacity and force generation are also features of aged and NMD-afflicted skeletal muscles. Since loss of SC number and function is also associated with aging and NMDs, it will be of interest to determine in these contexts the interrelationship between reduced integrity of Pax7+ SCs impaired NMJ regeneration and correlated skeletal muscle dysfunction.

22C57BL/6, Pax7CreERT2 (017763) Rosa26mTmG (007576) and Rosa26DTA (009669) mice were obtained from Jackson Laboratories (Bar Harbor, ME). Rosa26mTmG or Rosa26DTA mice were crossed with Pax7CreERT2 mice to generate Pax7CreER/+; Rosa26mTmG/+ (P7mTmG) or Pax7CreER/+; Rosa26DTA/+ (P7DTA) mice and control CreER negative (Ctrl) littermates. Transgenic mouse lines were used at 3–6 months of age. P7DTA and Ctrl mice were treated with (0.1 mg Tmx/g body weight) for 5 consecutive days I.P., 7 days after the first Tmx injection mice underwent SNT and sham surgeries. P7DTA and Ctrl mice were given additional Tmx 10 and 17 days after surgery. P7mTmG mice were similarly administered Tmx, however without additional Tmx injections after surgery. All animal procedures were conducted in accordance with institutional guidelines approved by the University Committee on Animal Recourses, University of Rochester Medical Center.

23Mice were anesthetized with intraperitoneal injections of ketamine (110 mg/kg) and xylazine (10 mg/kg). The hindquarter was then carefully shaved and depilation completed with generic Nair hair removal cream prior to skin cleansing with gauze. The skin was incised 1 mm posterior and parallel to the femur, and the biceps femoris was bluntly split to expose the sciatic nerve. 1–2 mm sciatic nerve was then transected 5 mm proximal to its trifurcation, followed with realignment of the distal and proximal nerve ends and closure of the muscle with wound clips (Autoclip, BD Clay Adams, Franklin Lakes, NJ). Mice were given analgesic (0.5–1.0 mg/kg buprenorphine) and allowed to recover on a heating pad. Sham surgery was performed on the contralateral leg where procedures were performed without nerve transection. At the designated times left and right hind limb muscles were collected. The muscles used for histology were incubated at 4°C overnight in 30% sucrose solution and frozen in dry ice-cooled isopentane.

24Flash-frozen muscles were sectioned at 10 μm (transverse) or 30 μm (longitudinal) and stored at −80°C. Sections were fixed for 3 min in 4% paraformaldehyde (PFA) (no PFA fixation for MyHC antibodies), and if needed, subjected to antigen retrieval: heating slides in citrate buffer (10 mM sodium citrate, pH 6.0) in a steamer (Oster 6.1 quart, model 5712, Racine, WI) for 15 min followed by cooling at room temperature for 2 min. Tissue sections were incubated with 0.2% Triton X-100 for 10 min, blocked in 10% normal goat serum (NGS, Jackson ImmunoResearch, West Grove, PA) 30 min at room temperature and stained with primary antibodies. If necessary (when mouse primary antibodies were used), sections were blocked in 3% affinipure Fab fragment goat anti-mouse IgG(H + L) (Jackson ImmunoResearch, West Grove, PA 115-007-003) with 2% NGS at room temperature for 1 hr. Primary antibodies were incubated at 4°C overnight or 2 hr at room temperature, and secondary antibodies were incubated for 1 hr at room temperature. DAPI (Invitrogen, Carlsbad, CA) staining was used to mark nuclei. All slides were mounted with Fluoromount-G (SouthernBiotech, Birmingham, AL).

25For H & E staining, flash-frozen sections were fixed for 3 min in 4% PFA, stained with Mayers Hematoxylin and Alcoholic Eosin Y, dehydrated, equilibrated with xylene and mounted using Cytoseal 60 (Richard-Allan Scientific, Kalamazoo, MI). For Sirius Red staining, a Picrosirius Red stain kit (Polysciences, Warrington, PA) was utilized. Briefly flash-frozen sections were fixed for 1 hr at 56°C in Bouin's fixative, washed in water, stained for 1 hr in Picrosirius Red, washed in 1 M HCl, dehydrated, equilibrated and mounted. Bright-field images were collected by a Zeiss Axioskop 40 microscope. Olympus VS110 virtual microscopy system was utilized for whole-slide scanning. Automatic quantification of MCT content was accomplished via VisioPharm software.

26Transverse sections and cells were imaged on a Zeiss Axio Observer A.1 microscope. Longitudinal sections were stained with SV2, Znp-1, 2H3, Btx and DAPI as described above and viewed with an Olympus Fluoview 1000 confocal microscope with 40X (for quantification) or 100X (for representative pictures) objectives at a 0.47 μm or 0.42 μm step size respectively. Amira software was used to analyze 3-D reconstructed NMJs for innervation analysis and to identify and enumerate post-synaptic myonuclei. Max-projection z-stack images of NMJs were generated with ImageJ software. The post-synaptic side was identified based on the entry of the terminal axon and as the concave side of the NMJ.

27For GFP localization, myofibers were purified by conventional collagenase digestion and trituration with fire polished glass pipets as previously described. Briefly, the EDL muscle was dissected, rinsed in Dulbecco's phosphate-buffered saline (PBS), put into a 1.5 ml eppendorf tube containing 1 ml 0.1% type I collagenase (Invitrogen) and 0.1% type II collagenase (Invitrogen) in Dulbecco's modified Eagles medium (DMEM, Sigma–Aldrich, St. Louis, MO), incubated in a shaker water bath at 37°C for 75 min and gently mixed by inversion periodically. Following digestion, the muscle was transferred to 100 mm × 15 mm plastic petri dishes containing 10 ml of plating media (10% horse serum in DMEM) using fire-polished-tip Pasteur pipettes. Under a stereo dissecting microscope, single myofibers were released by gently triturating the EDL with a series of modified Pasteur pipettes that varied in tip diameter to accommodate the progressive decrease in muscle trunk size. Inseparable fibers and debris were removed. Purified single myofibers were fixed with 4% PFA for 3 min, washed with PBS and transferred to 5 ml polystyrene cell collection tubes for GFP, Pax7 and Btx IF. For assessing single myofiber size and myonuclear number, muscles were fixed in 4% PFA for 48 hr prior to dissection and NaOH mediated digestion. Fixed muscles were incubated in 40% NaOH for 2 hr and agitated vigorously for 20 min. Released fibers were then washed in PBS and processed for DAPI staining.

28To assess cell proliferation C57BL/6 mice were fed BrdU (Sigma–Aldrich) (0.5 mg/ml supplemented with 5% sucrose) in drinking water after denervation. Muscles were collected 4 weeks after SNT and sectioned for Pax7, BrdU and Laminin IF.

29Whole EDL muscle contractility and force generation were analyzed using an ASI muscle contraction system (Aurora Scientific, Aurora, Canada) as described previously. Briefly, mice were anaesthetized and TA muscles removed. EDLs were then carefully isolated, adjusted to optimal length and stimulated at various frequencies to obtain absolute force values. To obtain specific force values, absolute force was normalized to pennation angle and cross-sectional area (determined by EDL weight and length). Muscle force was recorded and analyzed using Dynamic Muscle Control, Clamp fit and Graph Pad Prism software.

30Pax7 (mouse IgG1, 1:100, Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA), BrdU (rat, 1:250, Abcam ab6326, Cambridge, UK), Laminin (rat or rabbit, 1:1500, Sigma–Aldrich L0663 or L9393), GFP (rabbit, 1:400, Millipore AB3030P, Billerica, MA), F1.652 (mouse IgG1, 1:40, DSHB), A4.840 (mouse IgM, 1:40, DSHB), NCL-MHC (mouse IgG1, 1:100, DSHB), SC-71 (mouse IgG1, 1:40, DSHB), 6H1 (mouse IgM, 1:40, DSHB), BF-35 (mouse IgG1, 1:40, DSHB), BF-F3 (mouse IgG1, 1:40, DSHB), SV2 (synaptic vesicle protein-2, mouse IgG1, 1:100, DSHB), Znp-1 (synaptotagmin-2, mouse IgG1, 1:200, DSHB) and 2H3 (neurofilament, mouse IgG1, 1:200, DSHB), AlexaFluor 488-conjugated α-Bungarotoxin (1:1000, Life Technologies B-13422, Grand Island, NY), AlexaFluor 647-conjugated α-Bungarotoxin (1:1000, Life Technologies B-35450), AlexaFluor 594-conjugated goat anti-mouse IgG (1:1500, Life Technologies A-11032), AlexaFluor 594-conjugated goat anti-mouse IgG1 (1:1500, Life Technologies A-21125), AlexaFluor 488-conjugated goat anti-mouse IgM (1:1500, Life Technologies A-21042), AlexaFluor 488-conjugated goat anti-rat IgG (1:1500, Life Technologies A-11006), AlexaFluor 647-conjugated goat anti-rabbit (1:1500, Life Technologies A-21244).

31Results are presented as mean +SEM. Statistical significance was determined by Student's t-tests for simple comparison, one-way ANOVA and Bonferroni multiple comparisons test for multiple comparisons with Graph Pad Prism software. p < 0.05 was considered as statistically significant.